ARTICLE

https://doi.org/10.1038/s41467-021-22507-3 OPEN

Extracellular cap domain is an essential component

of the TRPV1 gating mechanism
Kirill D. Nadezhdin1,5, Arthur Neuberger1,5, Yury A. Nikolaev2, Lyle A. Murphy2, Elena O. Gracheva2,3,4,
Sviatoslav N. Bagriantsev 2 & Alexander I. Sobolevsky 1 ✉
1234567890():,;

Transient receptor potential (TRP) channels are polymodal molecular sensors involved in
numerous physiological processes and implicated in a variety of human diseases. Several
structures of the founding member of the TRP channel family, TRPV1, are available, all of
which were determined for the protein missing the N- and C-termini and the extracellular S5-
P-loop. Here, we present structures of the full-length thirteen-lined ground squirrel
TRPV1 solved by cryo-EM. Our structures resolve the extracellular cap domain formed by the
S5-P-loops and the C-terminus that wraps around the three-stranded β-sheet connecting
elements of the TRPV1 intracellular skirt. The cap domain forms a dome above the pore’s
extracellular entrance, with four portals leading to the ion conductance pathway. Deletion of
the cap increases the TRPV1 average conductance, reduces the open probability and affects
ion selectivity. Our data show that both the termini and the cap domain are critical deter-
minants of TRPV1 function.

1 Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA. 2 Department of Cellular and Molecular Physiology, Yale

University School of Medicine, New Haven, CT, USA. 3 Department of Neuroscience, Yale University School of Medicine, New Haven, CT, USA. 4 Program in
Cellular Neuroscience, Neurodegeneration and Repair, Yale University School of Medicine, New Haven, CT, USA. 5These authors contributed equally: Kirill D.
Nadezhdin, Arthur Neuberger. ✉email: as4005@cumc.columbia.edu

NATURE COMMUNICATIONS | (2021)12:2154 | https://doi.org/10.1038/s41467-021-22507-3 | www.nature.com/naturecommunications 1

ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22507-3

I
n mammals, the detection of high temperature is carried out
by terminal endings of primary afferent neurons expressing
thermosensitive ion channels of the transient receptor poten-
tial (TRP) family1. Several mammalian species have evolved the
ability to comfortably withstand high environmental heat for a
long period of time, which allowed them to inhabit otherwise
prohibitively hot ecological niches. Among these are small diurnal
rodents, including 13-lined ground squirrels (Ictidomys tride-
cemlineatus), who developed tolerance to environmental heat via
modification of the primary noxious heat sensor, TRPV1
(refs. 2–4). In contrast to TRPV1 orthologues from rat (rTRPV1)
and other mammals, ground squirrel TRPV1 (sqTRPV1) fails to
activate by heating in a physiologically relevant temperature
range, even though the channel is sensitive to chemical agonists,
such as acid and capsaicin5. The available structures of rTRPV1
revealed the overall architecture and key elements of the gating
machinery6–8, but all of these structures were solved for a trun-
cated construct that lacked the N- and C-termini, as well as the
extracellular S5-P-loop. In fact, these missing regions have been
implicated in the mechanism of temperature sensitivity9–18,
which remains poorly understood.
In this work, we study the full-length sqTRPV1 using cryo-
electron microscopy (cryo-EM) combined with electrophysiology
and mutagenesis to understand the general mechanisms of TRPV1
function. We show that the S5-P-loops form an extracellular cap
domain that shapes portals for ions to enter the central channel
pore and is critical for channel conductance, open probability
and ion selectivity. We also show that the C-terminus of the full-
length TRPV1 wraps around the three-stranded β-sheet making
numerous molecular interactions that strengthen intersubunit
interfaces and stabilize the intracellular skirt.
Results and discussion
Apo-state structure. We expressed full-length wild-type
sqTRPV1 (ref. 5) in HEK 293 cells and subjected the purified apo
protein to cryo-EM (Supplementary Fig. 1 and Supplementary
Table 1). The resulting structure (Fig. 1a–c) has a similar overall
shape and architecture, as the previously published structures of
truncated rTRPV1 (refs. 6–8; Supplementary Fig. 2). However, of
the three regions missing in rTRPV1—the N-terminus, the S5-P-
loop and the C-terminus—the latter two appear to fold into
structural elements in the context of full-length sqTRPV1. The C-
terminus of sqTRPV1 represents an extended polypeptide that
wraps around the three-stranded β-sheet (Fig. 1d), which con-
nects the ankyrin repeat domains of the neighbouring subunits to
form the typical for TRPV channels intracellular skirt. Electro-
static and hydrophobic interactions between the C-terminus, the
three-stranded β-sheet and the C-terminal hook of one subunit,
and the loops of ankyrin repeats AR2–5 of the neighbouring

a b
Cap

Out B
90° C
A C

In

C-terminus

B D A
D

90°

c d pre-S1
A AR5

E3 D
E2
D
E1

AR4
B
AR3

Hook
C AR2 N126
A AR1

Fig. 1 Structure of full-length sqTRPV1. a–c Side (a), top (b) and bottom (c) views of full-length sqTRPV1 structure with four subunits coloured differently,
the cap domain in blue and the C-terminus in pink. Dark blue sphere is a putative chloride ion. d Close-up view of the C-terminus with the residues
contributing to interactions with other domains shown in sticks. Asparagine N126, the serine substitution of which converts weakly temperature-sensitive
sqTRPV1 into highly temperature-sensitive rTRPV1-like channel, is labelled.

2 NATURE COMMUNICATIONS | (2021)12:2154 | https://doi.org/10.1038/s41467-021-22507-3 | www.nature.com/naturecommunications

NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22507-3
subunit make the intersubunit interfaces stronger. As a result, the
intracellular skirt of full-length sqTRPV1 is less dynamic than
that of truncated rTRPV1. Indeed, the entire skirt is well-resolved
in the cryo-EM density of full-length sqTRPV1, while density
for two peripheral of the six ankyrin repeats, as well as the
C-terminal hook domain preceding the third strand of the three-
stranded β-sheet in truncated rTRPV1 was missing6–8. Other
than these differences, apo-state structures of sqTRPV1 and
rTRPV1 are quite similar (Supplementary Fig. 2a, d), suggesting
that whenever the complete termini are present in rTRPV1, they
are likely to interact with the rest of the protein in the same way
as in sqTRPV1.
Similar to sqTRPV1, the extended C-terminus that wraps
around the three-stranded β-sheet has also been observed in cryo-
EM structures of other temperature-sensitive TRPV channels
TRPV2–3 (refs. 19–22). Compared to TRPV2–3, the C-terminus in
the full-length sqTRPV1, after wrapping around the β-sheet,
makes a U-turn and follows the edge of the skirt, providing
additional interdomain interactions (Fig. 1d). Unwrapping of the
C-terminus and binding of an otherwise unfolded region of the N-
terminus have been recently shown to accompany temperature-
induced opening of TRPV3 (ref. 23). Similarly, both N- and C-
termini may contribute to temperature gating of TRPV1.
Supporting this idea, the serine substitution of asparagine N126
converted weakly temperature-sensitive sqTRPV1 into highly
temperature-sensitive rTRPV1-like channel5. N126 is located at
the end of the AR1 helix that is in direct contact with AR2 helix
contacting the C-terminus (Fig. 1d). Thus, alteration of the
AR1–AR2 interaction is expected to have an immediate allosteric
effect on the binding of the C-terminus. In contrast, in
temperature-insensitive TRPV5–6, the C-terminus does not wrap
around the three-stranded β-sheet, and the N-terminal helix,
which pillars the corners of the intracellular skirt24,25, plays the
role of an intersubunit “glue”.
Extracellular cap. The S5-P-loops of the full-length sqTRPV1
assemble into an extracellular cap domain that forms a dome
above the pore extracellular entry (Fig. 1a, b). The cryo-EM
density for the cap is relatively weak and is completely missing for
residues 608–617. This can be explained by the dynamic nature
of the S5-P-loops and by possible spontaneous crosslinking of
cysteines C623, which in the fourfold symmetrical model of
sqTRPV1 come close together at the centre of the cap with 5.5 Å
distances between their Cα atoms. In case such crosslinking does
happen, the population of channels that contribute to cryo-EM
reconstruction is likely heterogeneous and includes particles with
non-symmetrical (one cysteine crosslink), twofold symme-
trical (two crosslinks) and fourfold symmetrical (no crosslinks)
cap domains.
Strong density in the middle of the cap, which we interpreted
as a chloride ion coordinated by arginines R624, represents a
likely barrier for cation conductance along the central axis of the
channel. On the other hand, four side portals in the dome lead to
the ion-conducting pore extracellular entrance (Fig. 2a, b). Each
portal is formed by the N-terminal part of the S5-P-loop of one
subunit and the C-terminal part of the S5-P-loop of the
neighbouring subunit, and contains numerous negatively charged
residues. Correspondingly, the overall negative charge of each
individual portal (Fig. 2b) creates a favourable environment for
passage of cations, and suggests a possible role of the cap domain
in TRPV1 conductance and ion selectivity. The missing residues
608–617 can potentially form a loop capable of occluding the side
portals, thus providing an additional way to regulate the ion
channel conductance.
NATURE COMMUNICATIONS | (2021)12:2154 | https://doi.org/10.1038/s41467-021-22507-3 | www.nature.com/naturecommunications
ARTICLE
To test the importance of the cap domain for the TRPV1
function, we generated a mutant channel lacking residues
606–628 (sqTRPV1 d606–628, Supplementary Fig. 3). Electro-
physiological recordings in Xenopus oocytes showed that
sqTRPV1 d606–628 lacks heat activation (Supplementary Fig. 4a),
similar to wild-type channel5. The amplitude histograms of cell-
attached single-channel recordings from HEK293T cells revealed
that deletion of the cap domain produced a significant increase in
single-channel conductance from 125 ± 4.9 pS for wild type to
174.6 ± 6.6 pS for the d606–628 mutant (P = 0.003, t test,
Fig. 2c–e)26. Compared to wild-type sqTRPV1, the d606–628
mutant exhibited flickering behaviour, in part due to the
significantly decreased mean open dwell time (Fig. 2f and
Supplementary Fig. 4b). In addition, we expressed sqTRPV1 wt
and sqTRPV1 d606–628 in Xenopus oocytes, and compared their
reversal potential, Erev, by recording currents in response to
different concentrations of capsaicin. At both 200 nM and 2 µM
of capsaicin, sqTRPV1 wt and sqTRPV1 d606–628 showed
significantly different values of Erev, suggesting that the deletion
of the S5-P-loop affects ion selectivity of sqTRPV1 (Supplemen-
tary Fig. 4c). Together, our functional data show that the cap
domain is an essential component of the TRPV1 gating
mechanism.
Lipids and ligands. The apo-state structure of the full-length
sqTRPV1 shows at least nine well-resolved densities of annular
lipids per channel subunit (Fig. 3a, b). The majority of densities
have a clear head-and-two-tails appearance and were modelled
with phosphatidylcholine. The exception is the lipid at site 1 or
the vanilloid binding site, which in structures of rTRPV1
accommodates agonists resiniferatoxin (RTX) and capsaicin, and
antagonist capsazepine6–8. Similar to the apo-state structure of
rTRPV1 (ref. 7), the vanilloid site in the apo-state structure of
sqTRPV1 contains density that is best fit by phosphatidylinositol
(PI, Fig. 3c). The head of PI is accommodated by polar and
charged residues of the linker domain (R411 and H412), the
S2–S3 loop (D511 and Y513), the S4–S5 linker (R559, E572 and
K573) and the TRP helix (Q702), while the PI tails face hydro-
phobic residues of S3 and S4 from one subunit, and S5 and S6
from the neighbouring subunit.
To get an insight into ligand binding to full-length TRPV1, we
determined structures of sqTRPV1 in complex with agonists RTX
and capsaicin, which both potently activate sqTRPV1, similar to
the rat orthologue (Supplementary Fig. 4d)5,27. In agreement with
the previous structures of rTRPV1, both RTX and capsaicin bind
to the vanilloid site and are coordinated by residues in S2–S3 loop,
S3, S4 and S4–S5 linker of one subunit, and S5 and S6 of the
neighbouring subunit (Fig. 3d, e and Supplementary Fig. 5). In the
apo state, the same residues contribute to the binding of PI, which
resides deeper in the vanilloid site towards the linker domain and
pushes away the side chain of tyrosine Y513. Binding of both RTX
and capsaicin results in shortening the distance between the
hydrogen bond-forming sidechains of T552 and E572, and
movement of the S4–S5 linker away from the pore (Supplemen-
tary Fig. 5b, c). This S4–S5 linker movement was proposed to be
coupled to S6 movement and pore opening28. While binding of PI,
RTX and capsaicin to full-length sqTRPV1 is nearly identical to
their binding to truncated rTRPV1, there is a notable difference in
the resulting architecture of the ion channel pore.
Ion channel pore. The pore of the full-length sqTRPV1 in the
apo and RTX-bound states (Fig. 4a, b) resembles the pore of the
truncated rTRPV1 in the apo state6–8 (Supplementary Fig. 2).
Measurements of the pore radius confirm that both the selectivity
3
ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22507-3

a R624 b Side portal

K605 R619
R619 K605
D603
E653 D603
D627 E650
D648 E650 D648 E602
E602 E638
S5 S6
S1 K641
P
P S4
S2 S3
S3
S5 S6
S4 S1 S2

c
sqTRPV1 wt sqTRPV1 d606-628
10 pA
50 ms

mV: mV:

+90 +80

+80 +70

+70 +60

+60 +50

10 pA
d 500 ms 20
20
Count (N x 100)
Count (N x 100)

10
10

0 0
0 10 20 30 0 10 20 30
Amplitude (pA) Amplitude (pA)
e f 8
20
1st level mean open time (ms)

sqTRPV1 wt
sqTRPV1 d606-628
**
15 6
Amplitude (pA)

10 4
174 pS

5 2

125 pS
0 0
0 50 100 150 wt d606-628
Voltage (mV)

filter and the lower gate of the channel are completely closed for capture the open-state structure of sqTRPV1, which can be
permeation of water and ions (Fig. 4d). Interestingly, the presence opened functionally without help of DKTX (Supplementary
of DKTX bound to rTRPV1 together with RTX leads to sig- Fig. 4d).
nificant pore widening and opening of the channel6–8 (Supple- The narrow constriction at the lower gate of sqTRPV1 in the
mentary Fig. 2e). It remains to be determined whether the open- apo and RTX-bound states is formed by the S6 residue I681,
state structure of rTRPV1 can be determined in the absence which is homologous to I679 that determines the narrowest
of DKTX. However, binding of RTX alone was not enough to diameter of the lower pore in all published structures of rTRPV1

4 NATURE COMMUNICATIONS | (2021)12:2154 | https://doi.org/10.1038/s41467-021-22507-3 | www.nature.com/naturecommunications

NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22507-3 ARTICLE

Fig. 2 Cap domain and its role in TRPV1 function. a Ribbon diagram of the sqTRPV1 extracellular portion with subunits in different colour and the cap
domain in blue. Charged residues are shown as sticks. b Surface view of the same region coloured by electrostatic potential with positively charged areas in
blue, negatively charged in red and neutral in white. c Representative traces from single-channel recording of spontaneous openings of wild-type sqTRPV1
and d606–628 mutant at different voltages in cell-attached patch from HEK293T cells at room temperature. Black arrows indicate the position of the
baseline current. Upward single-channel deflections represent outward current. d Amplitude histograms corresponding to the channel activity at 90 mV for
wild-type sqTRPV1 and d606–628 mutant. e Current–voltage relationship for wild-type sqTRPV1 and d606–628 mutant with its single-channel
conductances, fitted to the linear equation (wt, N = 5 cells; d606–628, N = 9 cells). Source data are provided as a Source data file. f Mean open dwell time
of wild-type sqTRPV1 and d606–628 mutant channels (at 70 mV). Each dot represents individual cell (N = 5 cells for each construct). Data are mean ±
SEM. **P = 0.0074, two-sided t test).

a Out
b 2 4
5
3
6 6

In
C
9

D 1
B 9
8

A
7

c L664 d S5 L664 e L664

S1 S4
S4 S4 F545
S5 F545 F545 A667
A667 F593 A667 S2 S1 S3 A548
S2 A548 S2 A548 M549
M549 M549 S5 F593 S6
F593 S6 RTX F589
S3 F589 N553 N553 T552
T552 T552
L671 L671 S6 L671
N553 F589 Cap
L517 PI L517 I575
L517
M516
M516 M516 Y513 I575
I575 L576 L576
Y513 S3 Y513
L576
E572
S5
S5 R559
R559
S1 E572 S5
E572
I705 K573 I705 R559 K573
S6 I705 D511 K573
Q702 D511 I698 Q702 I698
D511 S6 Q702
I698

L701
L701 L701 H412
H412 H412 R411 TRP helix
R411 TRP helix
R411 TRP helix

Fig. 3 Lipids and ligands. a Cryo-EM density for the apo-state full-length sqTRPV1, with the lipid densities coloured red or blue. b Close-up view of the
membrane region, with the molecules of lipid shown in sticks and numbered. c–e Close-up view of the vanilloid site (site 1 in b) in the apo-state structure
harbouring the molecule of phosphatidylinositol (c), RTX-bound (d) and capsaicin-bound (e) structures. The molecules of phosphatidylinositol, RTX and
capsaicin are shown as yellow, blue and green sticks, respectively, with the corresponding cryo-EM density shown in red.

(refs. 6–8). In contrast, the narrow constriction of the lower pore
in the capsaicin-bound structure of sqTRPV1 is formed by the S6
residue M684, while I681 faces away from the pore (Fig. 4c). This
change occurs because S6, which contains a central π-bulge in
other conformations of sqTRPV1 and rTRPV1, becomes entirely
α-helical in the capsaicin-bound structure of sqTRPV1. While the
α-helical S6 has not been observed in TRPV1 structures before6–8,
it has been predicted29 and has also been observed in structures of
other representatives of TRPV channels21,22,24,30–32. In fact, the
α-to-π transition in S6, which results in ~100° rotation of the
bottom portion of S6 and exposure of a completely different set of
residues to the channel pore, has been shown experimentally by
determining structures of TRPV6 (ref. 33), TRPV3 (refs. 21,22) and
TRPV2 (ref. 34) in different conformations. By solving structures
of sqTRPV1 in the apo, RTX-bound and capsaicin-bound states,
we now demonstrate experimentally that the α-to-π transition in
S6 can happen in TRPV1 and propose that this transition is an
essential feature of TRPV1 gating.
Measurements of the pore radius suggest that the ion channel
of sqTRPV1 in the capsaicin-bound state is in a closed non-
conducting conformation (Fig. 4d). The channel is too narrow to
conduct water or ions at both the selectivity filter and the lower
pore, where the narrow constriction is formed by M684. In
contrast, despite remaining non-conducting, the lower pore in the
capsaicin-bound structure of truncated rTRPV1 is dilated
compared to its apo-state structure6–8 (Supplementary Fig. 2f).
Whether the difference in the pore size and the presence of the α-
to-π transition in S6 originates from usage of different TRPV1
orthologs or from truncated versus full-length constructs of the
channel remains to be determined. The fact that the pore of
sqTRPV1 or rTRPV1 (refs. 6–8) remains closed, while the agonists
RTX or capsaicin are bound to the channel suggests that the

NATURE COMMUNICATIONS | (2021)12:2154 | https://doi.org/10.1038/s41467-021-22507-3 | www.nature.com/naturecommunications 5

ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22507-3

a b c d
50
M646 M646
M646 M646 M646 M646
P P Selectivity
P P S6 P G645 P S6 G645 Filter

Distance Along Pore (Å)

S6 G645 I644 40
I644 V669 I644 V669 S6
V669 S6 S6 T643
T643 T643 T672 T672 Central
Y673 Y673 T672
T672
S S L677
T672 T672
S S L677 Y673 D D Y673 30 cavity
L676 L676
L676 N678 L676 N678
L676 L677 L677
L680 I681 L680 I681 L680 L676
L680 N678 N678
I681 L680 I681
M684 M684 M684 M684 A682 20 Gate
A682 A682 A682
V688 V688 M684 M684
T687 T687 T687 T687 T687 T687
N689 N689
V688 10
V688
N689

Apo RTX Capsaicin 0

0 1 2 3 4 5 6 7
Pore Radius (Å)

Fig. 4 Ion channel pore. a–c Pore-forming domains in the apo (a), RTX-bound (b) and capsaicin-bound (c) states with the residues lining the pore shown
as sticks. Only two of four subunits are shown, with the front and back subunits omitted for clarity. The pore profiles are shown as space-filling models
(gray). The region that undergoes α-to-π transition in S6 is highlighted in pink. d The pore radius for the sqTRPV1 structures calculated using HOLE47. The
vertical dashed line denotes the radius of a water molecule, 1.4 Å.

channel resides in inactivated states. Future studies are required
to better understand the role of these conformations in the gating
mechanism of TRPV1.
Methods
Construct. The full-length 13-lined ground squirrel TRPV1 (GenBank
KU877439.1, residues 1–840)5 used in cryo-EM studies was cloned into a pEG
BacMam vector35, followed by thrombin cleavage site (LVPRG), enhanced green
fluorescent protein (eGFP) for monitoring during expression and a C-terminal
streptavidin affinity tag (WSHPQFEK) for purification.
Expression and purification. sqTRPV1 construct was expressed and purified as
previously described for mTRPV3 (refs. 22,23), with minor modifications. Bacmids
and baculoviruses were produced using a standard method35. Briefly, baculovirus
was made in Sf9 cells for ~72 h (Thermo Fisher Scientific, mycoplasma test
negative, GIBCO #12659017) and was added to the suspension adapted HEK 293
cells lacking N-acetyl-glucosaminyltransferase I (GnTI−, mycoplasma test negative,
ATCC #CRL-3022) that were maintained in Freestyle 293 media (Gibco-Life
Technologies #12338-018) supplemented with 2% FBS at 37 °C and 5% CO2.
Twenty-four hours after transduction, 10 mM sodium butyrate was added to
enhance protein expression, and the temperature was reduced to 30 °C. Seventy-
two hours after transduction, the cells were harvested by centrifugation at 5471 × g
for 15 min using a Sorvall Evolution RC centrifuge (Thermo Fisher Scientific),
washed in phosphate-buffered saline (pH 8.0), and pelleted by centrifugation at
3202 × g for 10 min using an Eppendorf 5810 centrifuge. The cell pellet was
resuspended in ice-cold lysis buffer, containing 20 mM Tris (pH 8.0), 150 mM
NaCl, 0.8 μM aprotinin, 4.3 μM leupeptin, 2 μM pepstatin A, 1 mM phe-
nylmethylsulfonyl fluoride and 1 mM β-mercaptoethanol (βME). Cells were sub-
sequently lysed using a Misonix Sonicator with a preset programme (six cycles of
15 s “on” at the amplitude of 8 followed by 15 s “off”; this programme was repeated
three times for optimal cell lysis) under constant stirring on ice. Unbroken cells and
cell debris were pelleted using an Eppendorf 5810 centrifuge at 3202 × g and 4 °C
for 10 min. The supernatant was subjected to ultracentrifugation in a Beckman
Coulter ultracentrifuge using a Beckman Coulter Type 45Ti rotor at 186,000 × g
and 4 °C for 1 h to pellet the membranes. The membrane pellet was mechanically
homogenized and solubilized in the lysis buffer supplemented with 20 mM n-
Dodecyl β-D-maltoside under stirring at 4 °C for 2 h. Insoluble material was
removed by ultracentrifugation for 40 min in a Beckman Coulter Type 45Ti rotor
at 186,000 × g, and the supernatant was added to strep resin and rotated for 14–16
h at 4 °C. Next, the resin was washed with ten column volumes of buffer containing
20 mM Tris (pH 8.0), 150 mM NaCl, 1 mM βME and 0.01% (w/v) glyco-diosgenin
(GDN), and the protein was eluted with the same buffer supplemented with
2.5 mM D-desthiobiotin. The eluted protein was concentrated using a 100 kDa
NMWL centrifugal filter (MilliporeSigma™ Amicon™) to 0.5 mg ml−1. The sample
was then digested with thrombin (1:200 mass ratio of thrombin to eluted protein)
for 1 h at 22 °C, and centrifuged in a Sorvall MTX 150 Micro-Ultracentrifuge
(Thermo Fisher Scientific) using a S100AT4 rotor for 30 min at 66,000 × g and 4 °C
before injecting into a size-exclusion chromatography (SEC) column. The protein
was further purified using a Superose™ 6 10/300 GL SEC column attached to an
AKTA FPLC (GE Healthcare) and equilibrated in 150 mM NaCl, 20 mM Tris,
1 mM βME and 0.01% GDN (pH 8.0). The tetrameric peak fractions were pooled
and concentrated using 100 kDa NMWL centrifugal filter (MilliporeSigma™ Ami-
con™) to 2.4–3.8 mg ml−1.
Cryo-EM sample preparation and data collection. Au/Au grids were prepared as
described in the literature36. Briefly, grids were prepared by first coating C-flat
(Protochips, Inc., Morrisville, NC) CF-1.2/1.3–2Au mesh holey carbon grids with
~60 nm gold, using an Edwards Auto 306 evaporator. Subsequently, an Ar/O2
plasma treatment (4 min, 50 watts, 35.0 sccm Ar, 11.5 sccm O2) was used to remove
the carbon with a Gatan Solarus (model 950) Advanced Plasma Cleaning System
(Gatan, Pleasanton, CA, USA). The grids were again plasma treated (H2/O2, 25 s,
10 watts, 6.4 sccm H2, 27.5 sccm O2) prior to sample application to make their
surfaces hydrophilic.
RTX and capsaicin were dissolved in dimethyl sulfoxide (DMSO) to a
concentration of 10 mM and 50 mM, respectively, and further diluted in buffer
containing 20 mM Tris (pH 8.0), 150 mM NaCl, 1 mM βME and 0.01% GDN to a
concentration of 1.5 mM. Ligands were added to protein 30 min before grid
preparation at the final concentration of 50 μM (RTX) and 300 μM (capsaicin).
Images of frozen-hydrated particles of sqTRPV1 (3.8 mg ml−1) in apo state
were collected on a Titan Krios transmission electron microscope (TEM; Thermo
Fisher Scientific) operating at 300 kV, and equipped with a post-column GIF
Quantum energy filter and a Gatan K3 Summit direct electron detection (DED)
camera (Gatan, Pleasanton, CA, USA) using Leginon. A total number of 11,191
micrographs were collected in counting mode with an image pixel size of 1.06 Å
and a defocus range of −0.8 to −2.5 µm, and energy filter slit of 30 eV. The total
dose of ~65 e− Å−2 was attained by using a dose rate of ~30 e− pixel−1 s−1 across
50 frames for 2.5 s total exposure time.
Images for frozen-hydrated particles of sqTRPV1 (2.4 mg ml−1) in the presence
of capsaicin were collected on a Titan Krios TEM (Thermo Fisher Scientific)
operating at 300 kV, and equipped with a post-column GIF Quantum energy filter
and a Gatan K3 Summit DED camera (Gatan, Pleasanton, CA, USA) using
Leginon. A total number of 9453 micrographs were collected in counting mode
with an image pixel size of 0.83 Å and a defocus range of −1.0 to −2.5 µm. The
total dose of ~58.5 e− Å−2 was attained by using a dose rate of ~16 e− pixel−1 s−1
across 50 frames for 2.5 s total exposure time.
Images of frozen-hydrated particles of sqTRPV1 in the presence of RTX were
collected on a Polara 300 kV TEM (FEI) with a Schottky field emission gun,
cartridge loading system and a Gatan K3 Summit DED camera (Gatan, Pleasanton,
CA, USA) using Leginon. A total number of 3253 micrographs were collected in
counting mode with an image pixel size of 0.95 Å and a defocus range of −1.5
to −3.5 µm. The total dose of ~70.8 e− Å−2 was attained by using a dose rate of
~16 e− pixel−1 s−1 across 40 frames for 4 s of total exposure time.
Image processing. All processing was completed in RELION37 and/or
cryoSPARC38 (Supplementary Table 1). The initial drift and beam-induced
motions were corrected using MotionCor2 (ref. 39) algorithm implemented in
RELION, and contrast transfer function (CTF) estimation was performed using
Gctf40. Following CTF estimation, micrographs were manually inspected and those
with outliers in defocus values, ice thickness and astigmatism, as well as micro-
graphs with lower predicted CTF-correlated resolution were excluded from the rest
of the processing pipeline (individually assessed for each parameter relative to
overall distribution; no set threshold). Initial set of particles was manually picked in
RELION and further classified into 100 2D classes to create templates for the
template-based picking in RELION that were used for the final round of picking.
Picked particles were further 2D- and 3D-classified in iterative classification using
RELION and/or cryoSPARC. The reported resolutions and local resolution pre-
dictions of the final maps were calculated in RELION and cryoSPARC, with the
resolution range estimated by the gold standard Fourier shell correlation 0.143

6 NATURE COMMUNICATIONS | (2021)12:2154 | https://doi.org/10.1038/s41467-021-22507-3 | www.nature.com/naturecommunications

NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22507-3
criterion41. EM density visualization was done in UCSF Chimera42 and UCSF
ChimeraX43.
Model building. To build models of TRPV1 in Coot44, we used the previously
published cryo-EM structures of TRPV1 as guides. The models were tested for
overfitting by shifting their coordinates by 0.5 Å (using shake) in Phenix, refining
each shaken model against a corresponding unfiltered half map, and generating
densities from the resulting models in Chimera. Structures were visualized, and
figures were prepared in UCSF Chimera, UCSF ChimeraX and Pymol45.
Patch-clamp electrophysiology. sqTRPV1-pMO5 or sqTRPV1 d606–628-pMO
constructs were co-transfected with pcDNA3-GFP into HEK293TΔPIEZO1 (ref. 46)
using Lipofectamine 3000 (Thermo Fisher), according to the manufacturer’s
instructions. The following day, cells were plated onto glass coverslips coated with
Matrigel Matrix (BD Bioscience) and analysed by voltage-clamp electrophysiology
within 24 h after plating. Bath solution contained (in mM): 140 KCl, 10 HEPES,
1 MgCl2, 10 glucose, pH 7.3 (pH adjusted with KOH) and the pipette solution
contained (in mM): 140 NaCl, 5 KCl, 10 HEPES, 1 EGTA, 1 MgCl2, pH 7.4
(pH adjusted with NaOH). To form the gigaohm seal, positive (+10 mmHg)
pressure was maintained inside the electrode while approaching the cell, and
released upon touching the cell membrane. Spontaneous single-channel events
were recorded in the cell-attached mode at room temperature at indicated voltages.
Single-channel openings are shown as upward deflections, representing outward
current. Currents were sampled at 25 kHz and filtered at 1 kHz. Single-channel
data was analysed using Clampfit 10.8. Mean single-channel amplitudes were
calculated by fitting the current-amplitude histograms with Gaussian curves. The
unitary conductance for sqTRPV1 was calculated by fitting the slope of the
current–voltage relationship to the linear equation. NPo and mean open time
values were calculated from 10 s recordings using Clampfit. RTX (Alomone, 2 mM
stock in ethanol) was applied by perfusion.
Oocyte electrophysiology. Defolliculated Xenopus laevis oocytes, stages V–VI
(Xenopus 1), were injected with sqTRPV1 mRNA (10 ng) and assayed 48–72 h
later by two-electrode voltage clamp in calcium-free ND96, containing (in mM):
3 KCl, 96 NaCl, 2 MgCl2 and 10 HEPES pH 7.4 (with NaOH), supplemented with
200 nM or 2 µM capsaicin. Currents were evoked by 200-ms voltage steps from
−100 to 50 mV, in 10 mV increments, from a holding potential of 0 mV, and
digitized at 2 kHz. The reversal potential was estimated from current–voltage
plots. Capsaicin (Sigma-Aldrich, 1 mM stock in DMSO) was applied by perfusion.
For temperature sensitivity experiments, currents were recorded in a gap-free
mode at a holding potential of −80 mV. Temperature was controlled using the
SC-20 in-line heater (Warner) and monitored by a thermistor placed in the
chamber near the oocyte.
Reporting summary. Further information on research design is available in the Nature
Research Reporting Summary linked to this article.
Data availability
All data needed to evaluate the conclusions of the paper are present in the paper and/or
the Supplementary Materials. Cryo-EM density maps have been deposited to the Electron
Microscopy Data Bank (EMDB) under the accession codes EMD-23491 (sqTRPV1 apo),
EMD-23492 (sqTRPV1 RTX), and EMD-23493 (sqTRPV1 capsaicin; see Supplementary
Table 1). The corresponding model coordinates have been deposited to the PDB under
accession numbers 7LQY (sqTRPV1 apo), 7LQZ (sqTRPV1 RTX), and 7LR0 (sqTRPV1
capsaicin; see Supplementary Table 1). All other data are available from the
corresponding author upon request. Source data are provided with this paper.
Received: 7 January 2021; Accepted: 18 March 2021;
References
1. Hoffstaetter, L. J., Bagriantsev, S. N. & Gracheva, E. O. TRPs et al.: a molecular
toolkit for thermosensory adaptations. Pflug. Arch. 470, 745–759 (2018).
2. Caterina, M. J. et al. The capsaicin receptor: a heat-activated ion channel in the
pain pathway. Nature 389, 816–824 (1997).
3. Caterina, M. J. et al. Impaired nociception and pain sensation in mice lacking
the capsaicin receptor. Science 288, 306–313 (2000).
4. Vandewauw, I. et al. A TRP channel trio mediates acute noxious heat sensing.
Nature 555, 662–666 (2018).
5. Laursen, W. J., Schneider, E. R., Merriman, D. K., Bagriantsev, S. N. &
Gracheva, E. O. Low-cost functional plasticity of TRPV1 supports heat
tolerance in squirrels and camels. Proc. Natl Acad. Sci. USA 113, 11342–11347
(2016).
NATURE COMMUNICATIONS | (2021)12:2154 | https://doi.org/10.1038/s41467-021-22507-3 | www.nature.com/naturecommunications
ARTICLE
6. Cao, E., Liao, M., Cheng, Y. & Julius, D. TRPV1 structures in distinct
conformations reveal activation mechanisms. Nature 504, 113–118 (2013).
7. Gao, Y., Cao, E., Julius, D. & Cheng, Y. TRPV1 structures in nanodiscs reveal
mechanisms of ligand and lipid action. Nature 534, 347–351 (2016).
8. Liao, M., Cao, E., Julius, D. & Cheng, Y. Structure of the TRPV1 ion channel
determined by electron cryo-microscopy. Nature 504, 107–112 (2013).
9. Vlachová, V. et al. Functional role of C-terminal cytoplasmic tail of rat
vanilloid receptor 1. J. Neurosci. 23, 1340–1350 (2003).
10. Brauchi, S., Orta, G., Salazar, M., Rosenmann, E. & Latorre, R. A hot-sensing
cold receptor: C-terminal domain determines thermosensation in transient
receptor potential channels. J. Neurosci. 26, 4835–4840 (2006).
11. Brauchi, S. et al. Dissection of the components for PIP2 activation and
thermosensation in TRP channels. Proc. Natl Acad. Sci. USA 104,
10246–10251 (2007).
12. Grandl, J. et al. Temperature-induced opening of TRPV1 ion channel is
stabilized by the pore domain. Nat. Neurosci. 13, 708–714 (2010).
13. Gracheva, E. O. et al. Ganglion-specific splicing of TRPV1 underlies infrared
sensation in vampire bats. Nature 476, 88–91 (2011).
14. Prescott, E. D. & Julius, D. A modular PIP2 binding site as a determinant of
capsaicin receptor sensitivity. Science 300, 1284–1288 (2003).
15. Yang, F., Cui, Y., Wang, K. & Zheng, J. Thermosensitive TRP channel pore
turret is part of the temperature activation pathway. Proc. Natl Acad. Sci. USA
107, 7083–7088 (2010).
16. Yao, J., Liu, B. & Qin, F. Modular thermal sensors in temperature-gated
transient receptor potential (TRP) channels. Proc. Natl Acad. Sci. USA 108,
11109–11114 (2011).
17. Zhang, F. et al. Heat activation is intrinsic to the pore domain of TRPV1. Proc.
Natl Acad. Sci. USA 115, E317–E324 (2018).
18. Clapham, D. E. & Miller, C. A thermodynamic framework for understanding
temperature sensing by transient receptor potential (TRP) channels. Proc. Natl
Acad. Sci. USA 108, 19492–19497 (2011).
19. Pumroy, R. A. et al. Molecular mechanism of TRPV2 channel modulation by
cannabidiol. Elife 8, e48792 (2019).
20. Zubcevic, L., Hsu, A. L., Borgnia, M. J. & Lee, S. Y. Symmetry transitions during
gating of the TRPV2 ion channel in lipid membranes. Elife 8, e45779 (2019).
21. Zubcevic, L. et al. Conformational ensemble of the human TRPV3 ion
channel. Nat. Commun. 9, 4773 (2018).
22. Singh, A. K., McGoldrick, L. L. & Sobolevsky, A. I. Structure and gating
mechanism of the transient receptor potential channel TRPV3. Nat. Struct.
Mol. Biol. 25, 805–813 (2018).
23. Singh, A. K. et al. Structural basis of temperature sensation by the TRP
channel TRPV3. Nat. Struct. Mol. Biol. 26, 994–998 (2019).
24. Saotome, K., Singh, A. K., Yelshanskaya, M. V. & Sobolevsky, A. I. Crystal
structure of the epithelial calcium channel TRPV6. Nature 534, 506–511
(2016).
25. Hughes, T. E. T. et al. Structural basis of TRPV5 channel inhibition by
econazole revealed by cryo-EM. Nat. Struct. Mol. Biol. 25, 53–60 (2018).
26. Yang, F. et al. An unorthodox mechanism underlying voltage sensitivity of
TRPV1 ion channel. Adv. Sci. 7, 2000575 (2020).
27. Elokely, K. et al. Understanding TRPV1 activation by ligands: Insights from
the binding modes of capsaicin and resiniferatoxin. Proc. Natl Acad. Sci. USA
113, E137–E145 (2016).
28. Vu, S., Singh, V., Wulff, H., Yarov-Yarovoy, V. & Zheng, J. New capsaicin
analogs as molecular rulers to define the permissive conformation of the
mouse TRPV1 ligand-binding pocket. Elife 9, e62039 (2020).
29. Kasimova, M. A. et al. A hypothetical molecular mechanism for TRPV1
activation that invokes rotation of an S6 asparagine. J. Gen. Physiol. 150,
1554–1566 (2018).
30. Zubcevic, L. et al. Cryo-electron microscopy structure of the TRPV2 ion
channel. Nat. Struct. Mol. Biol. 23, 180–186 (2016).
31. Huynh, K. W. et al. Structure of the full-length TRPV2 channel by cryo-EM.
Nat. Commun. 7, 11130 (2016).
32. Deng, Z. et al. Cryo-EM and X-ray structures of TRPV4 reveal insight into ion
permeation and gating mechanisms. Nat. Struct. Mol. Biol. 25, 252–260
(2018).
33. McGoldrick, L. L. et al. Opening of the human epithelial calcium channel
TRPV6. Nature 553, 233–237 (2018).
34. Dosey, T. L. et al. Structures of TRPV2 in distinct conformations provide
insight into role of the pore turret. Nat. Struct. Mol. Biol. 26, 40–49 (2019).
35. Goehring, A. et al. Screening and large-scale expression of membrane proteins
in mammalian cells for structural studies. Nat. Protoc. 9, 2574–2585 (2014).
36. Russo, C. J. & Passmore, L. A. Electron microscopy: ultrastable gold substrates
for electron cryomicroscopy. Science 346, 1377–1380 (2014).
37. Scheres, S. H. RELION: implementation of a Bayesian approach to cryo-EM
structure determination. J. Struct. Biol. 180, 519–530 (2012).
38. Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC:
algorithms for rapid unsupervised cryo-EM structure determination. Nat.
Methods 14, 290–296 (2017).
7
ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22507-3
39. Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced
motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332
(2017).
40. Zhang, K. Gctf: real-time CTF determination and correction. J. Struct. Biol.
193, 1–12 (2016).
41. Henderson, R. et al. Outcome of the first electron microscopy validation task
force meeting. Structure 20, 205–214 (2012).
42. Pettersen, E. F. et al. UCSF Chimera–a visualization system for exploratory
research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).
43. Pettersen, E. F. et al. UCSF ChimeraX: structure visualization for researchers,
educators, and developers. Protein Sci. 30, 70–82 (2021).
44. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics.
Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).
45. DeLano, W. L. The PyMOL Molecular Graphics System (DeLano Scientific,
2002).
46. Lukacs, V. et al. Impaired PIEZO1 function in patients with a novel autosomal
recessive congenital lymphatic dysplasia. Nat. Commun. 6, 8329 (2015).
47. Smart, O. S., Neduvelil, J. G., Wang, X., Wallace, B. A. & Samsom, M. S.
HOLE: a program for the analysis of the pore dimensions of ion channel
structural models. J. Mol. Graph. 14, 354–360 (1996).
Acknowledgements
Some of this work was performed at the Columbia University Cryo-Electron Microscopy
Center. The work was also performed at the National Center for CryoEM Access and
Training (NCCAT) and the Simons Electron Microscopy Center located at the New York
Structural Biology Center, supported by the NIH Common Fund Transformative High-
Resolution Cryo-Electron Microscopy programme (U24 GM129539), and by grants from
the Simons Foundation (SF349247) and NY State Assembly. E.O.G. was supported by
grants from NIH (1R01NS091300) and NSF (1754286). S.N.B. was supported by grants
from NIH (1R01NS097547) and NSF (1923127). A.I.S. was supported by the NIH (R01
CA206573, R01 NS083660, R01 NS107253) and NSF (1818086).
Author contributions
K.D.N. and A.N. made constructs, prepared protein samples, developed and optimized
cryo-EM grid preparation, and carried out cryo-EM data collection and processing.
8 NATURE COMMUNICATIONS | (2021)12:2154 | https://doi.org/10.1038/s41467-021-22507-3 | www.nature.com/naturecommunications
K.D.N., A.N. and A.I.S. analysed structural data. Y.A.N. performed single-channel and
inside-out patch-clamp electrophysiology. L.A.M. performed two-electrode voltage-
clamp recordings. K.D.N., A.N., E.O.G, S.N.B. and A.I.S. wrote the manuscript.
Competing interests
The authors declare no competing interests.
Additional information
Supplementary information The online version contains supplementary material
available at https://doi.org/10.1038/s41467-021-22507-3.
Correspondence and requests for materials should be addressed to A.I.S.
Peer review information Nature Communications thanks Makoto Tominaga, Jie Zheng,
and other, anonymous, reviewers for their contributions to the peer review of this work.
Reprints and permission information is available at http://www.nature.com/reprints
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
Open Access This article is licensed under a Creative Commons
Attribution 4.0 International License, which permits use, sharing,
adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative
Commons license, and indicate if changes were made. The images or other third party
material in this article are included in the article’s Creative Commons license, unless
indicated otherwise in a credit line to the material. If material is not included in the
article’s Creative Commons license and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from
the copyright holder. To view a copy of this license, visit http://creativecommons.org/
licenses/by/4.0/.
© The Author(s) 2021